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. 2024 Nov 28;13:RP95172. doi: 10.7554/eLife.95172

Figure 6. Subgroups of Foxp2-V1 interneurons defined by transcription factor (TF) expression at P5 and birthdate.

Figure 6.

(A) Otp (blue) and Foxp4 (white) expression in lineage-labeled Foxp2-V1s (EGFP, green) and non-Foxp2-V1s (tdT, red). The boxed area is shown at higher magnification with different color combinations for clarity. It shows that Foxp4-IR cells in the Foxp2-V1 population always expressed Otp (arrows). Quantification: 49.8–51.2% of Foxp2-V1s express Otp and 20.7–21.7% Foxp4 (n=2 mice each examined in 3 ventral horns in L4/5). Very few non-Foxp2-V1 cells express either TF (Otp: 1.3–4.2% and Foxp2: 0.5–2.9%). (B) Images of P5 spinal cords containing Foxp2-V1 lineage labeling (EGFP, omitted for clarity) and double immunolabeled for Otp/Foxp2, Foxp4/Foxp2, and Otp/Foxp4. (C) Quantification of Foxp2-V1 interneurons with different combinations of TF expression at P5. For each combination, the left panels show cell distributions, and the right graphs the percentage of Foxp2-V1s with each combination (n=3 mice each analyzed in 6 ventral horns). The data was analyzed with one-way ANOVAs followed by Bonferroni-corrected pairwise comparisons (statistical details in Supplementary file 1e, f, and g. ****p<0.0001; ***p<0.001; **p<0.01; *p<0.05). There are five main groups defined by TF expression patterns: Otp-Foxp2 (44%), Otp-Foxp2-Foxp4 (23%), Otp only (15%), Foxp2 only (9%), and no TF labeling (32% by subtracting all other groups). Some groups associate with specific locations: Otp-Foxp2 cells and the smaller proportion of Otp-only cells are located laterally; Otp-Foxp4-Foxp2 cells are located lateroventrally; medial cells either contain only Foxp2 or nothing; some dorsal cells are either Foxp2 only or do not express any of these TFs. (D) Example image of 5-ethynyl-2'-deoxyuridine (EdU) birthdating in En1Cre, Foxp2flpo, R26 RCE:dualGFP/Ai9 lsl-tdT dual-color mice pulse labeled at E11. Foxp2-V1s (green and yellow cells) are born between E10 and E12, with peak birthdate around E11 and after non-Foxp2 V1s (tdTomato only). The lower graph’s data is normalized to highlight the time of peak neurogenesis for each population (n=15.0 ± 4.1 ventral horns from one mouse per time point from E10 to E11 and 2 mice per time from E11.5 to E12.5; total 9 mice; bars show SEM). There is no difference between green (EGFP only) and the smaller population of yellow cells (EGFP and tdTomato). (E) EdU labeling in the Foxp2-V1 dual-color genetic model combined with Otp and Foxp4 antibody staining. (F) V1s expressing Foxp2, Otp, and Foxp4 at P5 are mostly born between E10.5 and E11.5 with neurogenesis time courses largely overlapping. The normalized plot indicates that peak neurogenesis for all three populations occurs at E11.5, although a marginally higher number of Otp V1 cells are born earlier (n=4 ventral horns from 3.27±1.34 mice per time point; error bars show SEM). Contour plots to the right show settling locations of Otp and Foxp4-IR populations born at each time point. (G) Schematic of the L4-5 ventral horn summarizing Foxp2-V1 subgroups according to location and combinatorial expression of Otp, Foxp4, and Foxp2 at P5. LMCD: dorsal lateral motor column; LMCV: ventral lateral motor column; MMC: medial motor column.